The concentrations of cortisol metabolites, 6-β-hydroxycortisol (6-OHF) and 17-hydroxycorticosteroids (17-OHCS), were measured in 24-hour urine specimens obtained before and during treatment of four healthy male volunteers with pentobarbital, 2 mg/kg, administered twice in a 12-hour period. There were definite increases in the excretion of 6-OHF in the urine of all four subjects after pentobarbital. Urinary 17-OHCS remained essentially unchanged. To determine whether this effect on cortisol metabolism was drug-specific, the study was repeated with another 13 volunteers using randomized double-blind conditions. There were higher concentrations of 6-OHF in the urine of subjects given the drug than in the urine of those given the placebo. The excretion of 17-OHCS in the pentobarbital-treated group was not significantly different from excretion in the control and placebo-treated groups. These results indicate that pentobarbital acutely increased the secretion of 6-OHF relative to total 17-OHCS, suggesting that pentobarbital is capable of stimulating hepatic microsomal hydroxylase activity. This raises the possibility that the urinary metabolites of cortisol may provide an indirect index of acute enzyme induction in man.